Composition for substrate surface modification and method using the same

ABSTRACT

The present invention provides a composition for substrate surface modification and a method using the same, and the composition for substrate surface modification is composed of a compound of the general formula structure shown in formula 1: 
     
       
         
         
             
             
         
       
         
         
           
             formula 1, wherein n 1  is an integer of 1 to 6, and R is a zwitterionic group. The composition for substrate surface modification uses water as a medium to perform modifying reaction over a substrate surface, and at the same time has biological modification characteristics, and abilities of immobilizing biomolecules and anti-biofouling.

FIELD OF THE INVENTION

The present invention relates to a composition for substrate surfacemodification, and more particularly to a compound containing azwitterionic functional group, and a method for modifying a substratesurface under the condition of using an aqueous solution as a medium.

BACKGROUND OF THE INVENTION

The material interface interacts with biomolecules to produce thechemical or physical interaction based on the chemical properties andphysical microstructures of a material, especially when a syntheticmaterial is placed in a biological environment. For most biomolecules,the biomolecules are usually non-selectively and naturally adsorbed onthe material surface. Therefore, the study of the interaction betweenbiomolecules and material interfaces is particularly important. Amongthem, the control material interface has anti-specific biomoleculeadsorption characteristics (hereinafter referred to as anti-biofouling),which is an indispensable part of the development of biomedicalmaterials.

The application of zwitterionic polymers in anti-biofouling technologyhas been developed for many years. So far, the application ofzwitterionic polymers in substrate surface modification still mainlyuses gas-phase deposition method (referred to as physical vapordeposition, chemical vapor deposition) or polymer polymerized reactionmethod, but the processes used in the two methods are complicated anddifficult to control.

When forming the zwitterionic polymer by the polymer polymerizedreaction method, the chemical structure for non-specific adsorption ofthe material has to be considered. The design principles include: thematerial must be hydrophilic; the overall chemical structure must beelectrically neutral; the graft density of the polymer on the surface ofthe material; and the physicochemical properties of the polymer bond.However, the polymer is difficult to be coated on the surface of thesubstrate homogeneously due to the poor hydrophilicity of the polymerbond and the high molecular weight of the polymer, which is the problemthat needs to be overcome at present.

SUMMARY OF THE INVENTION

In order to solve the above problems, a main object of the presentinvention is to provide a composition for substrate surface modificationand a method using the same, which utilize the stability andhydratability of a silatrane with a zwitterionic group for water toperform modifying reaction over a substrate surface with water as amedium.

Another main object of the present invention is to provide a method formodifying a substrate surface by using a free radical polymerizationmethod to prepare a silatrane with a zwitterionic group. Thus, it isable to save more time and easily controls in operation by the method.In accordance with the above objects, the present invention firstprovides a composition for substrate surface modification composed of acompound described in formula 1:

-   -   formula 1, wherein n₁ is an integer of 1 to 6, and R is a        zwitterionic group.

In a preferred embodiment of the present invention, the zwitterionicgroup is selected from the group consisting of the general formulasdescribed in formula 2-1 to formula 2-26:

-   -   wherein n is an integer of 1 to 3, and Q1 and Q2 can be the same        or each of a hydrogen atom, an alkyl group with carbon number of        1 to 6, a cycloalkane group with carbon number of 1 to 6, or an        aralkyl group with carbon number of 1 to 6.

The present invention further provides a method for modifying asubstrate surface, comprising: preparing a surface modificationsolution, providing a substrate to be surface-modified, and coating thesurface modification solution on a surface of the substrate to besurface-modified for reaction to modify the surface of the substrate.The above method, wherein steps of preparing the surface modificationsolution comprise: providing a 2-(dimethylamino) ethyl trimethoxy silaneas a reaction initiator; reacting the reaction initiator with a3-methyloxetane-2-one to obtain an intermediate product; purifying theintermediate product to obtain a purified intermediate product; adding atriethanol amine and a toluene to react with the purified intermediateproduct to obtain a final reactant; adding a dimethyl sulfoxide to thefinal reactant to prepare a standard solution; and diluting the standardsolution to form the surface modification solution.

In a preferred embodiment of the present invention, after coating thesurface modification solution on the substrate surface, furthercomprising steps of: activating the surface of the surface-modifiedsubstrate by using 1-ethyl-3-(3-dimethylamino propyl)carbodiimide (EDC)and N-hydroxy succinimide (NHS); adding a biomolecule to be modified tothe surface of the substrate for reaction; and performing a deactivatingreaction by adding a buffer solution to the surface of the substrate toobtain the surface-modified substrate by the biomolecule.

According to the above, the composition for substrate surfacemodification and the method for modifying the substrate surface of thepresent invention can be used on various substrates such as metals,polymers or glass in an aqueous solution environment. By using acovalent bond force between the zwitterionic group and the biomolecules,the biomolecules are closely attached to the substrate surface. Sincethe ability of the biomolecule being adsorbed on the substrate surfaceis correlated to the graft density of the zwitterionic group, thesurface of the substrate can be quickly modified and biologicallymodified, and has the ability of anti-biofouling.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is comparative graphs of the absorbance unit (AU) and thenormalized absorbance intensity (AI) for various examples;

FIG. 2 is a graph of linear regression of the analysis of the absorbanceof Example 2 and Comparative Example 2 measured by a spectrometeraccording to an embodiment of the present invention; and

FIG. 3 is a graph of linear regression of the analysis of the absorbanceof Example 3 measured by a spectrometer according to an embodiment ofthe present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In order to make the above and other objects, features and advantages ofthe present invention more comprehensible, a composition for substratesurface modification and a method using the same are described below,and related implementations and embodiments thereof are provided toillustrate the present invention and its efficacies specifically.

A main component of the composition for substrate surface modificationof the present invention is a silatrane with a zwitterionic group, andthe compound of the formula 1 is composed of a silatrane and azwitterionic group.

-   -   formula 1, wherein n₁ is an integer of 1 to 6, and R is a        zwitterionic group.

In the present invention, the silatrane is a tricyclic cage symmetricalstructure composed of a silicon nitride bond as a main axis, and thesilatrane has water vapor tolerance and low sensitivity to water so thatit is very suitable for surface bio-modification. On the contrary,silanes that have been traditionally and widely used in surface coatingtechnology of bio-sensing will cause problems of surface aggregation andunevenness because silane functional groups are easily hydrolyzed.

The zwitterionic group comprises at least an anionic group and acationic group. The anionic group includes a sulfo group, a carboxylgroup, a phosphate group or a phosphite ester group, and the cationicgroup can be a primary, secondary, tertiary or quaternary amino group, a5-membered heterocycle or a 6-membered heterocycle containing 1 oxygenatom or more than 1 nitrogen atom, for example, preferably animidazolyl, a pyrazolyl, or a pyridyl; but is not limited thereto. Thezwitterionic group can also contain other chemical groups. The anionicgroup and the cationic group contained in the zwitterionic group are anelectronegative functional group and an electropositive functionalgroup, respectively. When they are in an equal proportion, they areelectrically neutral and can be ionically paired with the substratesurface, which is referred to a non-covalent bond, and form a covalentbond with the amino group contained in the biomolecules. Further, astrong interaction of the covalent bond and the non-covalent bond isformed on the substrate surface, so that the biomolecule is closelyattached to the substrate surface. The mentioned substrate generallyrefers to various materials such as metals, polymers, or glass, and isnot limited thereto.

In a preferred embodiment of the present invention, the zwitterionicgroup is selected from the group consisting of the general formulasdescribed in formula 2-1 to formula 2-26:

-   -   wherein n is an integer of 1 to 3, and Q1 and Q2 can be the same        or each of a hydrogen atom, an alkyl group with carbon number of        1 to 6, a cycloalkane group with carbon number of 1 to 6, or an        aralkyl group with carbon number of 1 to 6.

According to the above composition for substrate surface modification,the present invention simultaneously provides a method of using acomposition for substrate surface modification, and preferably using thecomposition for substrate surface modification of the present inventionin an environment of water. An environment of dichloromethane or ethanolis also able to use the composition for substrate surface modificationof the present invention. The main reason is that the zwitterionic groupof the silatrane contained in the composition for substrate surfacemodification of the present invention is a hydrophilic end, and thezwitterionic group will produce a hydrogen to bond with the water or theaqueous solution of a polar solvent such as dichloromethane or ethanol;the composition for substrate surface modification is preferably graftedonto the substrate surface by water in order to adhere to the substratesurface, and various substrates such as metals, polymers or glass can beapplied. Thus, this method can reduce the consumption of organicsolvents and avoid damage to the surface of the substrate.

According to the above composition for substrate surface modification,the present invention simultaneously provides a method for modifying asubstrate surface, which sequentially comprises steps of (S1) preparinga surface modification solution, and (S2) performing surfacemodification on the substrate. The foregoing steps are described indetail below.

(S1) Preparing a Surface Modification Solution

The surface modification solution of the present invention contains thesilatrane with the zwitterionic group, which is a polymer formed byring-opening polymerization. Procedure steps for preparing the surfacemodification solution are represented as the following reaction formula1 to reaction formula 5, comprising step a as shown in the reactionformula 1: providing a (dimethylamino)ethyl trimethoxysilane as areaction initiator as shown in formula 3, and reacting the reactioninitiator with a 4-methyloxetan-2-one as shown in formula 4 to obtain anintermediate product. After a tertiary amino group of the2-(dimethylamino) ethyl trimethoxy silane nucleophilically attacks themonomer of 4-methyloxetan-2-one to perform ring-opening, the pure wateris added as a terminator to terminate the reaction to obtain theintermediate product as shown in formula 5; step b as shown in thereaction formula 2: purifying the intermediate product by extraction toobtain a purified intermediate product as shown in formula 5; step c asshown in the reaction formula 3: adding a triethanol amine and a tolueneas shown in formula 6 to react with the purified intermediate product toobtain a final reactant as shown in formula 7; step d as shown in thereaction formula 4: adding a dimethyl sulfoxide to the final reactant toprepare a standard solution, wherein the dimethyl sulfoxide is a polarsolvent and is miscible with water to stably store the final reactant;and finally, step e as shown in the reaction formula 5: diluting thestandard solution with dichloromethane, ethanol or pure water to formthe surface modification solution.

(S2) Performing Surface Modification on the Substrate

Reacting the substrate to be surface-modified (for example, varioussubstrates such as metals, polymers, or glass, but not limited thereto)with the surface modification solution, by immersing the substrate inthe surface modification solution to react, or coating the surfacemodification solution on the substrate surface to react. Since thesurface modification solution has strong hydratability and is able tobond with the substrate surface to produce hydroxyl interaction, thehydroxyl provides a receptor for intermolecular hydrogen bonding, andthe substrate is modified to have a hydrophilic surface to resistadsorption of biomolecules. Thus, the surface-modified substrate has theability of anti-biofouling.

In one embodiment, the present invention further provides a method formodifying a substrate surface with abilities of both bio-modificationand anti-biofouling. The method sequentially comprises steps of (S1)preparing a surface modification solution; (S2) performing surfacemodification on the substrate; (S3) activating the surface of thesurface-modified substrate; (S4) adding biomolecules to the substratesurface for reaction; and (S5) performing a deactivating reaction. Step(S1) and step (S2) are performed as described above, and will not bedescribed again. The following describes the consequent steps.

(S3) Activating the Surface of the Surface-Modified Substrate

Activating the surface-modified substrate by using1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS), then rinsing the substrate surface with water toremove excess EDC/NHS from the environment.

(S4) Adding Biomolecules to the Substrate Surface for Reaction

Adding biomolecules to the activated substrate. Since the activatedsubstrate has a hydrophobic surface after activation, an irreversiblebiomolecule adsorption layer film is formed when the activated substratecontacts with biomolecules, such as proteins, antibodies, cells,bacteria, etc. As the hydrophobic interaction between the hydrophobicend of the biomolecule structure and the substrate surface is increased,the biomolecule will be more strongly adsorbed on the substrate surface.Then, washing away the excess biomolecules with water.

(S5) Performing a Deactivating Reaction

Performing a deactivating reaction on the substrate surface adsorbedwith the biomolecules with a buffer solution to obtain a substrate witha surface modified by the biomolecules. For example, the buffer solutionis selected from a phosphate buffer solution, a sodium bicarbonatebuffer solution, a carbonate buffer solution, preferably a phosphatebuffer solution. When performing the deactivating reaction, the hydroxylgroup forms a hydrogen bond with the water molecule to generate a stablehydration layer, so that biomolecule layer on the biomolecule-modifiedsubstrate retains sufficient hydration layer to prevent other types ofbiomolecules from getting close and being adsorbed. Therefore, themethod for modifying the substrate surface of the present invention hasabilities of both bio-modification and anti-biofouling.

The details of the various examples of the present invention areprovided below to more clearly illustrate the present invention, but thepresent invention is not limited to the following examples.

Preparation Example of the Surface Modification Solution:

2-(dimethylamino) ethyl trimethoxy silane and 4-methyloxetan-2-one areplaced in a beaker containing acetonitrile to react for 12 hours toobtain an intermediate product, and the intermediate product is purifiedby extraction at room temperature. Adding triethanol amine and reactingin a toluene environment for 6 hours to obtain a final reactant. Then,dimethyl sulfoxide is added to prepare a standard solution with aconcentration of 1M to be stored, and the standard solution is diluted1,000 times in water to prepare the surface modification solution with afinal concentration of 1 mM.

Preparation Example of the Surface-Modified Substrate:

The surface modification solution is dip-coated on a plasticcolorimetric tray to react for 30 minutes, and then the plasticcolorimetric tray is rinsed with deionized water and alcohol, and driedby cold air to obtain the surface-modified substrate.

Anti-Biofouling Test:

Example 1: A bovine serum albumin solution with a high concentration (1mg/ml) is prepared and added to the surface-modified substrate for 2minutes. Then, the surface-modified substrate is washed with deionizedwater. The absorbance density (OD) at a wavelength of 590 nm is measuredwith a spectrometer after adding a coomassie blue reagent.

Please refer to FIG. 1. The left graph of FIG. 1 is an absorbancespectra among Example 1 according to one embodiment of the presentinvention, Reference Example 1, and Comparative Example 1 which aremeasured by a spectrometer. In the left graph of FIG. 1, the thick solidline represents the absorbance unit (AU) of Example 1, the dash linerepresents the absorbance unit of Reference Example 1 and the thin solidline represents the absorbance unit of Comparative Example 1 (Controlexperiment). Further, the right graph of FIG. 1 is a normalizedabsorbance intensity (ΔI) of Example 1 according to one embodiment ofthe present invention and Comparative Example 1.

In the left graph of FIG. 1, Comparative Example 1 (thin solid line) isa substrate without surface modification which shows the absorption ofthe bovine serum albumin Example 1 (thick solid line) is a substratewith surface modification according to one embodiment of the presentinvention, which shows no absorption of the bovine serum albumin. Theabsorbance curve of the Bradford reagent in Reference Example 1 (dashline) is used as a comparison baseline to demonstrate the smudgeresistance to the bovine serum albumin.

In the right graph of FIG. 1, the normalized absorbance intensity at awavelength of 590 nm of the surface-modified substrate is 0 in Example1, indicating no bovine serum albumin is adsorbed; and the normalizedabsorbance intensity of Comparative Example 1 with unmodified substrateis 0.04, indicating the bovine serum albumin is adsorbed. Therefore, theplastic colorimetric tray comprising the surface-modified substrates isprovided with the effect of anti-fouling against the bovine serumalbumin

Identification Test of Bio-Specificity:

Example 2: The Surface Modification Solution with Five differentconcentrations are dip-coated on five 96-well plastic plates to reactfor 30 minutes, then the plastic plates are rinsed with deionized waterand alcohol, and dried by cold air to obtain surface-modified plasticplates. The five surface-modified plastic plates are activated andreacted with EDC/NHS for 7 minutes, then the excess EDC/NHS is washedaway with deionized water. The poly Q amyloid (Heterogeneous Huntingtinprotein) are respectively added into the five surface-modified plasticplates to react for 15 minutes. The excess protein is washed away withdeionized water, and deactivating reaction is carried out to react for 2minutes by adding a phosphate buffer solution with a pH of 9. Then, goldnanoparticles with specificity for the poly Q amyloid are synthesizedand added onto the five plastic plates to react for 15 minutes. Theabsorbance of each of the plastic plates in Example 2 at a wavelength of520 nm is measured with a spectrometer.

Comparative Example 2: The five surface-modified plastic plates areobtained in the similar manner as in Example 2. The only differencerelies on that Comparative Example 2 replaces poly Q amyloid withamyloid-β (Aβ₁₋₄₀). The absorbance of each of the plastic plates in theComparative Example 2 at a wavelength of 520 nm is measured with aspectrometer.

Accordingly, the absorbance data of the surface-modified plastic platesadded with poly Q amyloid and Aβ₁₋₄₀ are respectively obtained from theExample 2 and Comparative Example 2, and the absorbance data of thesurface-modified plastic plates are applied to run a linear regressionanalysis respectively. FIG. 2 is a graph of linear regression of theabsorbance between Example 2 and Comparative Example 2 measured by aspectrometer. It can be known that the absorbance unit of thesurface-modified plastic plates added with the poly Q amyloid variesdepending on the concentrations of the surface-modified plastic plates.However, the absorbance unit of the surface-modified plastic platesadded with the Aβ₁₋₄₀ are not changed with the concentrations of thesurface-modified plastic plates. Thus, the surface-modified plasticplates in Example 2 have an identification ability of bio-specificity tothe poly Q amyloid. That is, the concentration of adsorbed poly-Qamyloid is correlated to the concentration of the surface modificationon the plastic plate. The absorbance shows a good linear relationshipwith the concentrations, which means that the detection limit is clearlyknown, and the surface-modified plastic plates comprising in Example 2is provided with high detection sensitivity.

Test of Specificity of Immunoreaction:

Example 3: Five surface-modified 96-well plastic plates are obtained inthe same manner as in Example 2. The surface-modified plastic plates areactivated and reacted by EDC/NHS for 7 minutes, then the excess EDC/NHSis washed away with deionized water, and 0.01 ppm of an anti-mouseantibody is added to react for 15 minutes. The excess anti-mouseantibody is washed away with deionized water, and deactivating reactionis carried out for 2 minutes by adding a phosphate buffer solution witha pH of 9 to complete modification of the surfaces of the plastic platesby the anti-mouse antibody. Then, the five plastic plates modified withthe anti-mouse antibody (used as an antibody) are added with mouseantibody of five different concentrations to react for 15 minutes, thelogarithmic concentrations of the mouse antibody (used as an antigen)are respectively 6.3, 6.0, 5.7, 5.4, 5.1. Further, a coomassie bluereagent is added to measure the absorbance each of the plastic plates ata wavelength of 595 nm with a spectrometer.

Five sets of absorbance data corresponding to different antigenconcentrations are obtained, and linear regression is performed as shownin FIG. 3. FIG. 3 is a graph of linear regression of the analysis of theabsorbance of Example 3 measured by a spectrometer, and the antigenconcentrations show a good linear relationship with the absorbance Thus,the plastic plates comprising the surface-modified substrates in Example3 is able to sense the specific immunoreaction of the antibodies to theantigens, which can be applied to bio sensing and biomedical materials.

In summary, the composition for substrate surface modification of thepresent invention allows the substrate surface being modified by water,so that the substrate will not be contaminated by organic solvents. Atthe same time, the substrate surface has biological modificationcharacteristics, the ability to immobilize biomolecules, andanti-biofouling characteristics, such as anti-protein adsorptioncontrol, anti-blood coagulation control, anti-tissue adhesion control,antibacterial control, etc.; Besides, after activation by EDC/NHS, thesubstrate surface can be biologically modified, so that the substratesurface has a high compatibility with biomolecules, a highidentification ability of bio-specificity, and can sense immunoreaction,which is considerably suitable for using in biomedical materials for,such as biomimetic engineering, bio-inspiration, and biological sensing.

What is claimed is:
 1. A method for modifying a substrate surface,comprising: preparing a surface modification solution, comprising:providing a 2-(dimethylamino) ethyl trimethoxy silane as a reactioninitiator; reacting the reaction initiator with a 4-methyloxetan-2-oneto obtain an intermediate product; purifying the intermediate product toobtain a purified intermediate product; adding a triethanol amine and atoluene to react with the purified intermediate product to obtain afinal reactant; adding a dimethyl sulfoxide to the final reactant toprepare a standard solution; and diluting the standard solution to formthe surface modification solution; providing a substrate to besurface-modified; and coating the surface modification solution on asurface of the substrate to be surface-modified for reaction to modifythe surface of the substrate.
 2. The method for modifying the substratesurface as claimed in claim 1, wherein the reaction initiator is reactedwith the 4-methyloxetan-2-one in an acetonitrile solution.
 3. The methodfor modifying the substrate surface as claimed in claim 1, wherein thestep of diluting the standard solution comprises using dichloromethane,ethanol or pure water to dilute.
 4. The method for modifying thesubstrate surface as claimed in claim 1, wherein the substrate is ametal, a polymer or a glass.
 5. The method for modifying the substratesurface as claimed in claim 1, wherein after coating the surfacemodification solution on the surface of the substrate, the methodfurther comprises steps of: activating the surface of thesurface-modified substrate using1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS); adding a modifying biomolecule to the surface of thesubstrate for reaction; and performing a deactivating reaction by addinga buffer solution to the surface of the substrate to obtain thesurface-modified substrate by the biomolecule.